1. Field of the Invention
The present invention relates to genetic sequences and proteins useful in the regulation of genes, particularly in the regulation of .kappa.B-containing genes.
2. Discussion of the Background
A twice-repeated 11-base-pair sequence, termed .kappa.B, which is known to exist in association with the enhancer of the immunoglobulin .kappa. light chain (Sen et al, Cell (1986) 46:705, Sen et al, Cell (1986) 47:729) is present in the control elements of numerous genes, including both vital and cellular genes. NF-.kappa.B, a regulatory element originally described as a transcription factor that recognizes the .kappa.B element of the immunoglobulin (Ig) .kappa. light chain enhancer and which is known to be associated with .kappa. light chain expression, has been found in a variety of vital enhancers, including SV40, CMV, HIV-2 and SIV. .kappa.B-related sites have been found in association with several cellular genes, including class I and II MHC genes, IL-2, IL-2 receptor (IL-2R.alpha.), serum amyloid A, .beta.-2 micro-globulin, .beta.-interferon, tumor necrosis factor, and T-cell receptor genes.
A .kappa.B-like site contributes functionally to IL-2-dependent gene expression and may also regulate IL-2R.alpha. transcription. .kappa.B is also found among the regulatory elements upstream of the HIV enhancer. This site was initially identified as a positive regulatory element recognized by DNA binding proteins found to be present in activated, but not resting, T cells. Mutation of the .kappa.B sites abolished inducibility of the HIV enhancer in T leukemia cells. A link between the .kappa.B site and the tat-I gene was also suggested by the synergistic response of the HIV enhancer to tat-I and NF-.kappa.B-mediated stimulation.
Another .kappa.B-like site was found upstream of the class I MHC gene. Although this site competes for binding to NF-.kappa.B, a DNA binding protein distinct from NF-.kappa.B has been identified in MEL and HeLa cells. Baldwin et al, Proc. Nat. Acad. Sci. (USA) (1988) 85:723. This protein, termed H2TF1, was detected in nuclear extracts containing no detectable NF-.kappa.B binding activity and had a different apparent molecular weight as measured by UV-cross-linking analysis. Another protein, designated KBF1, also recognized this site and may be related in part to NF-.kappa.B. Israel et al, Proc. Nat. Acad. Sci. (USA) (1987) 84:2653, Yanno et al, EMBO J (1987) 6:3317.
Although these proteins recognize .kappa.B-like sites, their relationship to the IL-2R .kappa.B binding protein(s) and NF-.kappa.B is unknown. Similarly, the HIVen86 protein is likely to be distinct from NF-.kappa.B since it has a higher apparent molecular weight. Franza et al, Nature (1987) 330:391. Its relationship to the IL-2R .kappa.B binding protein, which has been designated R.kappa.B (NF-r.kappa.B), and its role in mediating transactivation dependent on these .kappa.B-like sites is also not yet understood. Although the evidence is indirect, it appears that HIVen86 also differs from R.kappa.B, which has a higher apparent molecular weight of 100 kD.
While extensive sequence similarity to .kappa.B is found among enhancers associated with several genes (Lung et al, Nature (1988) 333:776), a second characteristic shared by these sites is their ability to respond to transactivation by the tax.sub.1 gene of HTLV-1. At the same time, several lines of evidence suggest differences among .kappa.B binding proteins. For example, nuclear extracts from MEL cells display an H2TF1 binding activity in the absence of detectable NF-.kappa.B binding. Competition studies showed that both the IL-2R.kappa.B and .kappa.B sites compete less efficiently than H2TF1 for binding to the site, consistent with previous studies suggesting that a distinct binding protein binds to the H2TF1 site.
Analysis of binding proteins by UV-cross-linking and SDS-page has also revealed multiple radiolabeled complexes, further implicating multiple proteins in .kappa.B binding. By UV-cross-linking, several specific complexes have been detected, including proteins of molecular weights of ca. 160, ca. 90, ca. 75, and ca. 50 kD. There is no evidence for differential regulation of these proteins by different NF-.kappa.B stimulants since PMA, TNF-.alpha., and IL-1 induce the same set of complexes. The complex of ca. 50 kD is consistent with previous report of NF-.kappa.B and/or .kappa.BF1. Although a .kappa.B binding protein of 86 kD HIVen86A, has also been identified by two-dimensional gel electrophoresis, it is unclear whether the ca. 90 kD protein represents this protein. Both HIVen86 and R.kappa.B show no increase in binding following cellular activation. The 160 kD complex has not been previously described.
In summary, at least seven .kappa.B binding proteins have been defined to date by either mobility shift analysis, UV-crosslinking, or protein purification. The molecular weight of these proteins range in size from 48 to greater than 300 kD. The various .kappa.B binding proteins that have been reported are indicated in Table 1 below, together with their relative specificities for the canonical .kappa.B, the class I MHC, and the IL-2R.kappa.B sites.
TABLE 1 ______________________________________ Summary of .kappa.B Binding Proteins Protein cDNA Name Specificity (kD) (kb) ______________________________________ NF-.kappa.B/KBFI .kappa.B = MHC = IL-2R.kappa.B 50 4 H2TF-1 MHC &gt; .kappa.B &gt; IL-2R.kappa.B 110 -- EBP-1 MHC = .kappa.B 60 -- HIVen86 .kappa.B = IL-2R.kappa.B 86 -- MBP-1 MHC &gt; .kappa.B &gt; IL-2R.kappa.B .about.300 9.5 R.kappa.B (IL-2R.kappa.B) IL-2R.kappa.B &gt; .kappa.B &gt; MHC 95 5.5 ______________________________________
Of these, the cDNAs encoding three of these proteins have been isolated. These cDNAs differ from one another by primary sequence and react with mRNAs of distinct size by northern blot. Taken together, these data indicate that multiple proteins could bind to a set of .kappa.B-related sites. Thus, there is a family of .kappa.B binding proteins, which, unlike some DNA binding proteins (e.g., c-jun), do not appear to be members of a related multigene family.
The .kappa.B sequence is known to exist in association with the regulatory elements of various viral and cellular genes. In light of the obvious interest in regulating gene expression there is accordingly a need for a factor useful in the regulation of .kappa.B-containing genes.